Accepted Article Preview: Published ahead of advance online publication Transcriptome analysis in blood cells from children reveals potential early biomarkers of metabolic alterations JSa´nchez, C Pico´, W Ahrens, R Foraita, A Fraterman, L A Moreno, P Russo, A Siani, A Palou, on behalf of the IDEFICS Consortium Cite this article as: JSa´nchez, C Pico´, W Ahrens, R Foraita, A Fraterman, L A Moreno, P Russo, A Siani, A Palou, on behalf of the IDEFICS Consortium, Transcriptome analysis in blood cells from children reveals potential early biomarkers of metabolic alterations, International Journal of Obesity accepted article preview 6 June 2017; doi: 10.1038/ijo.2017.132. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply. Received 18 January 2017; revised 6 April 2017; accepted 23 May 2017; Accepted article preview online 6 June 2017 © 2017 Macmillan Publishers Limited. All rights reserved. Transcriptome analysis in blood cells from children reveals potential early biomarkers of metabolic alterations Juana Sánchez1, Catalina Picó1, Wolfgang Ahrens2,3, Ronja Foraita2, Arno Fraterman4, Luis A. Moreno5, Paola Russo6, Alfonso Siani6, Andreu Palou1 on behalf of the IDEFICS Consortium. 1Laboratory of Molecular Biology, Nutrition and Biotechnology (Nutrigenomics), University of the Balearic Islands (UIB) and CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Palma de Mallorca, Spain. 2Leibniz Institute for Prevention Research and Epidemiology - BIPS, Bremen, Germany. 3Faculty of Mathematics and Computer Science, University of Bremen (UNIHB), Bremen, Germany. 4MVZ Eberhard & Partner Laboratoriumsmedizin, Dortmund, Germany. 5GENUD (Growth, Exercise, Nutrition and Development) Research Group, Faculty of Health Sciences, Universidad de Zaragoza, Instituto Agroalimentario de Aragón (IA2), Instituto de Investigación Sanitaria Aragón (IIS Aragón), Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Zaragoza, Spain. 6Unit of Epidemiology and Population Genetics, Institute of Food Sciences, National Research Council, Avellino, Italy. Running title: Early biomarkers of metabolic alterations Corresponding author and person to whom reprint should be addressed: Prof. Catalina Picó. Universitat de les Illes Balears, Biología Molecular, Nutrición y Biotecnología (Nutrigenómica). Edifici Mateu Orfila. Carretera de Valldemossa Km 7.5, 07122-Palma de Mallorca, Spain. Telephone: +34 971173070; Fax: +34 971173426. e-mail: [email protected] Disclosure statement: The authors have nothing to disclose. 1 © 2017 Macmillan Publishers Limited. All rights reserved. Abstract Objectives. The development of effective strategies to prevent childhood obesity and its comorbidities requires new, reliable early biomarkers. Here, we aimed to identify in peripheral blood cells (PBCs) potential transcript-based biomarkers of unhealthy metabolic profile associated to overweight/obesity in children. Methods. We performed a whole-genome microarray analysis in blood cells to identify genes differentially expressed between overweight and normal weight children to obtain novel transcript-based biomarkers predictive of metabolic complications. Results. The most significant enriched pathway of differentially expressed genes was related to oxidative phosphorylation, for which most of genes were down-regulated in overweight versus normal weight children. Other genes were involved in carbohydrate metabolism/glucose homeostasis or in lipid metabolism (e.g. TCF7L2, ADRB3, LIPE, GIPR), revealing plausible mechanisms according to existing biological knowledge. A set of differentially expressed genes was identified to discriminate in overweight children those with high or low triglyceride levels. Conclusion. Functional microarray analysis has revealed a set of potential blood-cell transcript- based biomarkers that may be a useful approach for early identification of children with higher predisposition to obesity-related metabolic alterations. 2 © 2017 Macmillan Publishers Limited. All rights reserved. Introduction Obesity is nowadays considered one of the main public health problems, affecting not only adults but also children. WHO estimated that 42 million children under the age of 5 were overweight or obese in 2013 (1). What is even more worrying is the greater probability of an obese child to maintain obesity into adulthood. Moreover, early onset of obesity is associated with an increased incidence of co-morbidities, such as type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular diseases and metabolic syndrome (2, 3). Knowing that obesity is influenced by several genetic, environmental and behavioural factors, and that the success of treatment is limited, prevention of obesity at early ages becomes a major need and should be one of the main focuses of attention. Thus, early diagnosis seems important for management and prevention of childhood obesity. The identification of biomarkers of predisposition to obesity-associated metabolic alterations could aid present/current strategies in decreasing in a more effective manner obesity associated co-morbidities, by targeting the underlying processes. Transcriptome analysis in peripheral blood cells (PBCs), considering whole peripheral blood or a subpopulation of white blood cells, the so-called peripheral blood mononuclear cells (PBMCs), has been proposed as a research strategy to identify new biomarkers and candidate- genes for a number of diseases based on differentially expressed mRNA profiles (4-7). PBCs have the advantage of being easy to collect, unlike invasive biopsies. Moreover, gene expression in these cells may reflect the responses of internal organs, such as adipose tissue and liver, and thereby have been proposed as a source of biomarkers of health and disease (8-10). More specifically, PBCs have been proposed as an appropriate method for studying the cardiovascular system (6), or as a source of transcript-based biomarkers for very early stages of acute coronary disease (5). In addition, the suitability of blood cells as a potential source for biomarkers of metabolic adaptations to food intake and body weight maintenance has been reported both in animal (8, 9, 11) and human studies (12, 13). Besides, in murine models, expression levels of selected genes in PBCs have been proposed as predictive biomarkers of a healthy or distorted metabolic state due to interventions during the perinatal period (14, 15). 3 © 2017 Macmillan Publishers Limited. All rights reserved. In the present study, a whole genome microarray analysis was performed in PBCs to identify genes differentially expressed between overweight and normal weight children, using a cross- sectional design, in order to derive novel transcript-based biomarkers that could be predictive of metabolic complications in early life. These biomarkers could be helpful for identifying particularly those obese children who are at risk of metabolic complications, hence allowing early interventions in a more effective way. Materials and Methods Participants IDEFICS is a large European multi-centre study on childhood obesity (details in (16)). Using a cross-sectional design, whole-genome microarray analysis has been performed in PBCs from a subgroup of 32 individuals with normal weight (17) and overweight (15), belonging to the Spanish IDEFICS cohort. Children included in the analysis were randomly selected within normal weight and overweight individuals and matched by sex. BMI categories were defined according to Cole (17). The age range was 4.7-8.0 years. Blood sampling and processing for gene expression analysis For each participant, a total of 2.5 mL peripheral blood was collected under fasting conditions into PAXgene vacutainer tubes (Qiagen, Izasa-Barcelona, Spain) via antecubital fossa venipuncture. Total RNA was isolated using the PAXgene blood RNA kit according to the manufacturer’s instructions (Qiagen) and as previously described (13). Microarray processing From each sample, 80 ng of RNA was reverse transcribed to complementary DNA (cDNA) using the Agilent Low Input Quick Amp Labeling kit (Agilent Technologies, Inc., CA, USA) according to the manufacturer’s protocol. Then, half of the cDNA sample (10 µl) was used for the linear amplification of RNA and labelling with cyanine-3 (Cy3) or Cy5. Transcription and labelling were carried out at 40 °C for 2 h. The labelled and amplified cRNA samples were purified using Qiagen Rneasy MiniSpin columns (Qiagen, Madrid, Spain). The incorporation of 4 © 2017 Macmillan Publishers Limited. All rights reserved. dyes and cRNA concentration was measured using the NanoDrop ND 1000 spectrophotometer (NanoDrop Techonologies, Ins., Wilmington, DE). Then, 825 ng of cRNA labelled with Cy5 from each sample and 825 ng of Cy3 pool was hybridized on 4x44K G4845A human whole genome Agilent microarrays (Agilent Technologies, Inc., Santa Clara, CA, USA) for 17 h at 65 °C in hybridization chambers in an oven rotating at 10 rpm (Agilent Technologies). After hybridization, the arrays were washed with "GE wash buffer 2" for 1 min at 37 °C, followed by acetonitrile for 10 s at room temperature, and finally with a solution